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1 Full Title: Emergence timing and voltinism of phantom midges, Chaoborus spp.,
2 in the UK.
3 Short Title: Chaoborus spp. emergence and voltinism.
4 Authors: R. Cockroft 1,W.R. Jenkins 2, A. Irwin 3, S. Norman2 and K.C. Brown4
5 1 AgroChemex Environmental Ltd., Aldhams Farm, Dead Lane, Manningtree, Essex, CO11 2NF,
6 U.K.,
7 2 Ridgeway Eco, Didcot, Oxfordshire, UK.
8 3 Independent Consultant, Norwich, U.K.
9 4 BrownEnvironmental, Haga, 61433 Söderköping, Sweden
10 Corresponding author: E-mail: [email protected]
11
12 This work was supported by funding from:
13 Adama Makhteshim Ltd., P.O. Box 60, Beer-Sheva, 8410001, Israel.
14 The sponsor did not influence the study design; the collection, analysis and interpretation of data or
15 the writing of the report.
16
17
18 Key words: aquatic invertebrates, Chaoborus, emergence timing, voltinism
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19 Abstract
20 After introduction of overwintered fourth instar larvae (2027 in total), emergence timing of adult
21 Chaoborus spp. (Diptera: Chaoboridae) was investigated in four outdoor freshwater microcosms in
22 the UK in 2017. Adults started emerging on 13 April and emergence reached a peak on 2 May. The
23 majority of emergence was completed by 3 June. Emergence rates for each microcosm ranged from
24 51.4% to 66.2% with a mean of 60.9%. The great majority of emerged adults were C. obscuripes
25 (99.68%). Males appeared to emerge slightly earlier than females. The results indicated that for
26 overwintered C. obscuripes larvae, the adults emerged en masse in spring (rather than emerging
27 gradually over the course of spring and summer). In a separate experiment at the same location, the
28 number of Chaoborus spp. life-cycles occurring per year was determined using six replicate groups of
29 microcosms, each group containing four microcosms. Each microcosm contained 200 L of water and
30 was enclosed within a ‘pop-up’ frame covered with ‘insect-proof’ mesh (1 mm2 aperture). The first
31 microcosm in each group was ‘seeded’ with egg rafts (first generation) of Chaoborus spp. Following
32 adult emergence, as soon as the first egg rafts were laid in each microcosm these were removed and
33 transferred to the second microcosm in that group, and so on. The larvae sampled from the second and
34 subsequent generations in the microcosms were all C. crystallinus. C. crystallinus produced up to four
35 discrete generations within the experimental period, and life-cycle times from egg-to-egg ranged from
36 14 days (replicate group 5, first generation) to 56 days (replicate 3, second generation). These two
37 experiments, indicated that i) adult C. obscuripes arising from overwintered larvae emerged en masse
38 in the spring, and ii) up to four generations of C. crystallinus occurred; i.e. C. crystallinus exhibited a
39 multi-voltine life history under the temperate conditions of this UK study.
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41 Introduction
42 The precise duration of the life-cycle of Chaoborus spp. does not appear to have been clearly defined
43 in published studies. Since the larval stages are known to be extremely sensitive to the effects of
44 insecticides (1) and size-dependent sensitivity can play an important role in the survival and recovery
45 of natural populations, the duration and timing of the life cycle has implications for the interpretation
46 of how natural populations recover from exposure to stressors.
47
48 The Chaoboridae are Diptera with aquatic larvae that live as pelagic predators, feeding on a wide
49 range of prey including copepods, Cladocera, rotifers, chironomids, mosquito larvae and other
50 chaoborids. There are three genera of Chaoboridae in northern and central Europe; Chaoborus,
51 Mochlonyx and Cryophilia, all of which have four aquatic larval instars (2, 3, 4). Chaoborus species
52 are widely distributed throughout Europe and of the six species that are known C. flavicans, C.
53 obscuripes and C. crystallinus are the most common, the most abundant, and the most studied.
54 Pelagic third and fourth instar larvae of some species, such as C. flavicans, have adapted to co-exist
55 with fish in larger water bodies and lakes. C. flavicans exhibits diel vertical migration to sediment
56 where they burrow during the daytime to avoid predators and migrate upwards at night to feed. In
57 shallower water, or where fish are absent, they may be entirely pelagic (5). Populations of C.
58 crystallinus predominantly occur in shallow water bodies without fish and are mostly pelagic,
59 although larvae have been found in sediment (5). Females of C. crystallinus are able to detect the
60 presence of fish kairomones in water (6) and so can avoid depositing their eggs in water bodies
61 containing fish.
62
63 Larger natural or anthropogenic water bodies that do not contain fish have been found to be
64 dominated by C. obscuripes (7, 8). Habitat preferences for the more common European species of
65 Chaoboridae are summarised in Table 1.
66
67
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68 Table 1: Habitat preferences for the larvae of European Chaoboridae
Species Location and Habitat
Larvae found in puddles, springs, pools, wells, tree holes and in temporary Mochlonyx velutinus or permanent pools.
Chaoborus flavicans* Lakes most often but also ponds.
Chaoborus crystallinus* Shallow ponds without fish, shallow ephemeral water bodies.
Man-made waters bodies without fish, small and shallow waters, also Chaoborus obscuripes* found in deeper lakes.
Chaoborus pallidus* Shaded portion of pools.
69 * overwinter as instar IV larvae in permanent water bodies
70 Chaoboridae are holometabolous and larvae develop through four instars and then pupate. Mochlonyx,
71 Cryophilia and Chaoborus nyblaei overwinter in the egg stage which is resistant to desiccation whilst
72 other temperate species overwinter as fourth instar larvae and pupate between April to June,
73 depending on temperature.
74
75 Mating and oviposition take place a few hours after emergence and in some species (e.g. C. flavicans)
76 eggs can be held at the surface by a surrounding jelly. C. crystallinus lays 200-300 eggs (8) arranged
77 in the form of floating discs to form a raft. The duration of the egg stage is temperature-dependent and
78 for C. crystallinus can range from 190-200 hours at 10 ºC and between 37-50 hours at 20 ºC (2). First
79 and second instar larvae of C. crystallinus develop rapidly over a few weeks whereas the
80 developmental periods of the third and fourth instar larvae are considerably longer. First and second
81 instar larvae are positively phototactic (9, 2) at first and stay in the upper layer of water (epilimnion),
82 which is warmer and has more oxygen. The later instars are generally found deeper in the water
83 column where they feed on zooplankton.
84
85 Chaoborus larvae are very tolerant of a wide range of unfavourable environmental conditions. C.
86 flavicans can withstand periods of up to 18 days without oxygen when the surface could not be
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87 reached and 70 days with access to the surface (2). Larvae of the same species can have variable life
88 spans (10) depending on environmental conditions and the structure of the community. Life cycles
89 can therefore be univoltine (predominantly in Europe) bi-voltine (high temperatures in Europe) or
90 multi-voltine in Japan (11).
91
92 The duration of the pupal stage is also temperature dependent and for Chaoborus crystallinus this can
93 range from 2-4 days at 20 ºC, between 10-13 days at 10 ºC and 30 days at 5 ºC. In Central Europe,
94 there appears to be a very pronounced emergence period for Chaoborus and Mochlonyx between
95 April and May with a second, less pronounced emergence from the end of July to October (10).
96 Females emerge from pupae and mate almost immediately, before their genitalia harden. Male
97 swarming behaviour is commonplace and some species have been shown to be attracted to light.
98 Imagines have no resting periods and live for at most ten days during which they do not feed (6, 9).
99
100 The presence of larvae throughout the year has led to the conclusion in published literature that
101 Chaoborus spp. are univoltine in temperate conditions. In central Europe, C. crystallinus was
102 considered to be univoltine, although possibly bi-voltine in hot summers (9, 12 -16 ). Verberk et al
103 (16) considered the dispersal strategy of Chaoborus crystallinus to be uni/bi-voltine and typical of
104 those species that have a long period of juvenile development. Life cycle strategies of Chaoborus
105 spp. reported in the literature are summarised in Table 2.
106
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108 Table 2: Number of generations per year for Chaoborus spp. from published literature
Species Number of generations
C. flavicans Univoltine (8, 18, 19, 20)
Univoltine or bivoltine (17)
C. crystallinus Univoltine (2, 8, 9, 13, 15, 16, 21)
Univoltine and possibly bivoltine in hot summers (12)
Multi-voltine (22, 23)
C. obscuripes Multi-voltine (24)
Univoltine (25)
109
110 Berendonk and Spitze (12) state in their introduction: ‘Chaoborus crystallinus is univoltine in Central
111 Europe although it may go through two generations in exceptionally hot summers.’. However, no
112 citation to support this statement is given in that paper. The Chaoborus found in microcosm studies
113 conducted in central Europe appear to be mostly C. crystallinus, as deduced by Janz et al. (23), who
114 analysed data collected from 19 microcosm studies conducted over 14 years at the University of
115 Munich, Germany. In that study, 1st and 2nd instar larvae were present from mid-April to early
116 October, 3rd instar larvae from early-May to October/November. Larvae overwintered as 4th instar and
117 these were present during the entire study. Pupae were found from early-April to the end of August.
118 The number of egg-laying peaks was used to indicate the number of generations of C. crystallinus in
119 that study (the first at the end of April/early May, the second in June and the third at the end of July
120 beginning of August). Janz et al concluded that there were three generations of C. crystallinus each
121 year.
122
123 As shown in Table 2, publications up to 2008 have considered C. crystallinus to be univoltine. On the
124 other hand, the two most recent papers (both published in 2016) are unequivocal in the conclusion
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125 that C. crystallinus is multi-voltine (in temperate conditions). We would surmise that the disparity
126 between the earlier and most recent articles is that the conclusions of the latter are based on empirical
127 evidence, whereas the statements on voltinism in the other papers appear to originate from
128 an assumption in earlier publications. The empirical evidence in (23) and (23) is essentially the
129 observed prevalence of the various C. crystallinus life-stages over time in outdoor microcosms. The
130 experiment described in this publication is a refinement of this approach, directly tracking the
131 progress of successive generations. The difference is that this new work excludes the confounding
132 factor of egg deposition by adults which have emerged from other water bodies. This was achieved
133 by the enclosing microcosms in ‘tents’ made of ‘insect-proof’ netting. The only C.
134 crystallinus inoculants were egg rafts placed by the experimenters. These rafts came from the
135 previous (also enclosed) generation. This could be described as a ‘temporal chain’, each link in the
136 chain being an artificial transfer of egg rafts from one enclosed microcosm to the next.
137
138 C. crystallinus is considered to be highly sensitive to the effects of certain insecticides and has been
139 used in individual based models to predict their potential effects and recovery of aquatic invertebrates
140 (26). An evaluation of the number of generations of C. crystallinus per year would be relevant for
141 understanding the recovery potential of Chaoborus spp. populations in freshwater systems following
142 possible reduction by pesticides.
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144 Materials and Methods
145
146 Two separate trials were conducted at AgroChemex Environmental Ltd., Aldhams Farm Research
147 Station in Essex, U.K. (Grid Reference TM 099 305) in 2017, one to investigate the emergence timing
148 of Chaoborus spp. adults from an overwintered cohort of 4th instar larvae and one to determine how
149 many generations per year could occur in the UK.
150
151 Microcosms of the same design were used in both trials. Each microcosm consisted of a circular
152 polypropylene tank approximately 0.8 m in diameter and 0.6 m deep (with a volume of 227 litres),
153 sunk into turfed ground to a depth of approximately 50 cm. 20 litres of washed sharp sand was added
154 and each microcosm was filled to a depth of approximately 50 cm (the same level as surrounding soil)
155 with approximately 200 L of freshly-drawn borehole water. Approximately 10 litres of water-
156 saturated lake sediment were poured in and allowed to settle in an even layer over the sand base and
157 each unit was covered with insect-proof mesh (1 mm2 aperture) to prevent the entry of aquatic flies,
158 particularly Chaoborus spp. An oxygenating submerged aquatic macrophyte (Elodea canadensis,
159 sourced from Envigo, Eye Research Laboratory, Suffolk, UK) was loosely planted in each microcosm
160 to occupy an area of approximately ¼ of the sediment surface. The plant material was rinsed
161 thoroughly prior to introduction into the systems to remove any invertebrates.
162
163 Populations of zooplankton (typically comprising rotifers, copepods, daphniids to provide a food
164 source for Chaoborus larvae) and detritus-feeding benthic invertebrates (e.g. Asellus and Gammarus
165 to facilitate natural recycling of nutrients) were sourced from a natural pond on the site at Aldhams
166 Farm Research Station and added to each microcosm. A handful of alder (Alnus glutinosa) leaves was
167 also added to each microcosm to provide a substrate for benthic invertebrates. Alder leaves had
168 originally been collected from Fen Alder Carr (a local nature reserve established in 1982), Suffolk,
169 UK, and then dried and stored. The alder leaves were soaked for >7 days in clean borehole water and
170 roughly shredded with scissors prior to addition to the microcosms.
171
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172 Active microcosms in both trials were monitored weekly for temperature, pH, dissolved oxygen and
173 conductivity using a Hach HQ40d portable multimeter . Water temperature in one unit was monitored
174 with readings every 30 minutes, using a calibrated data logger. Additionally, the water temperature in
175 an unused, unenclosed microcosm was also monitored continually from June onwards, to allow a
176 comparison of temperatures in enclosed and unenclosed systems. Climatic conditions on the
177 microcosm site were recorded throughout the study using a Davis Vantage Pro2 Plus field weather
178 station, situated approximately 100 metres from the study area.
179
180 Emergence timing.
181 This study was conducted to determine whether the first spring emergence of adult Chaoborus from
182 overwintered 4th instar larvae took place over a defined period, or was protracted, with adults
183 emerging at intervals throughout the year.
184
185 Four microcosms established between February and March 2017 were used for this study. Populations
186 of Chaoborus spp. were established on 22 March 2017 in each microcosm by the addition of
187 approximately 500 4th instar larvae, obtained from an untreated field reservoir at Envigo, Wooley
188 Road, Alconbury, Huntingdon, Cambridgeshire, UK. Chaoborus were collected using a sweep net
189 and transferred to a covered holding vessel containing water from the source reservoir for
190 transportation to the field site. Each container held larvae collected from several sweeps of the water
191 column, just below the water surface. On arrival, larvae were held outdoors in their original containers
192 with loosely fitting covers. On the day of initiation, groups of approximately 50 larvae were
193 transferred into a tray, counted and then added to one of the replicate microcosms. This process was
194 repeated until the four microcosms contained 502, 503, 510 and 512 larvae respectively. Before the
195 start of the study, the microcosms were covered with insect-proof netting to prevent colonisation by
196 the local populations of Chaoborus. Plate 1 shows the caged microcosms and Plate 2 shows an adult
197 male Chaoborus spp.
198
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199 Plate 1. Microcosms with mesh-covered frames
200 Plate 2. Adult male Chaoborus crystallinus male. Photo Eugène Vandebeulque 201
202 The emergence of adult Chaoborus sp. was monitored daily in the four microcosms by collecting
203 emerged insects seen on the walls of the enclosure or on the surface of the ground around the
204 microcosm using a vacuum sampler. Collected insects were preserved in 70% alcohol and stored for
205 subsequent identification to species level. The water surface was inspected daily for the presence of
206 egg rafts deposited by emerged females that had mated before sampling. Any egg rafts observed were
207 removed as soon as practically possible to prevent the introduction of fresh larvae to the microcosms.
208
209 Voltinism in Chaoborus
210 This experimental system consisted of six replicates each of four individual microcosms established
211 between February and March 2017 at the field site. An additional set of four microcosms was
212 established to provide the initial egg rafts and these were covered with a pop-up frame and insect
213 proof mesh (Popadome, Harrod Horticultural, Lowestoft, UK).
214
215 Each of the four microcosms established for the production of egg rafts were initiated with
216 approximately 500 4th instar Chaoborus spp. larvae from the same source as used to initiate the
217 emergence experiment and covered with insect-proof netting. Following the emergence of adult
218 Chaoborus spp., these egg generation microcosms were regularly monitored for the presence of egg
219 rafts on the water surface. When egg rafts were found they were transferred to the first of the four
220 microcosms in each replicate set. The production of egg rafts in the egg generation microcosms was
221 monitored until no more egg rafts were required. Each of the first of the four microcosms in each
222 replicate set containing egg rafts was inspected at least three times each week for the appearance and
223 development of larvae, pupation, emergence of adults and deposition of egg rafts. The presence of
224 larvae and their approximate instar (estimated by eye) together with the presence or absence of pupae
225 and emerged adults was recorded by inserting a 19 cm diameter white disc attached to a rod to
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226 provide contrast for assessing both at the surface (early-instar larvae) and at depth (late instar larvae
227 and pupae) and visual inspection of the enclosure mesh (adults). The date and numbers of any egg
228 rafts produced were also recorded.
229 Emerged adult insects were allowed to remain within the enclosure, reproduce and deposit egg rafts
230 on the water surface. These eggs were then collected and added to the second microcosm of each
231 replicate set to initiate populations. Established microcosms were inspected three times per week until
232 the end of September when the monitoring frequency was reduced to once per week. The presence or
233 absence of each life stage of Chaoborus was recorded in each active microcosm. Where present, an
234 approximation of the size range of larvae visible was recorded, mainly to facilitate monitoring of egg
235 hatching success and the rate of development, to ensure that critical development stages were not
236 missed. Once adult emergence had been observed, at each subsequent assessment, the water surface
237 was inspected for the presence of egg rafts deposited by emerged females. The observation of the
238 first deposition of egg rafts was recorded and those egg rafts used to initiate the next sequential unit
239 within the replicate. Subsequently, additional egg rafts produced within the active units were also
240 transferred to supplement the next unit’s population, until it was considered that no more were
241 required.
242 On three occasions, once in July and twice in October, samples of late-instar larvae were taken and
243 preserved in 70% alcohol for identification to species level. In the July sampling, only three
244 microcosms contained larvae considered sufficiently developed for identification and ten larvae were
245 sampled from each. In October, where larvae were abundant, approximately 30 were sampled and if
246 fewer than this were seen, all larvae which could be captured were preserved.
247
248 The process of monitoring the appearance and development of larvae, presence of pupae, the
249 emergence of adults and deposition of egg rafts was repeated for the second, third and fourth
250 generations when applicable. In each case, the date from the first appearance of egg rafts in any
251 generation was used to estimate the duration of the life cycle time from egg-to-egg of each generation.
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252 Larvae were sampled and identified to species level to determine the population composition in each
253 microcosm.
254 Results
255 Emergence timing
256 Of the 1228 emergent adults, all but four were identified as C. obscuripes. Counts of the numbers of
257 emerged adult male and female C. obscuripes in each of the four microcosms are shown in Figure 1.
258
259 Figure 1: Emergence timing of male and female Chaoborus obscuripes
260
261 Three specimens of C. crystallinus were found, the first (in Replicate 2) on 24 April 2017 (Day 25
262 post-initiation) and one each on 01 and 05 September 2017 (Days 154 and 158, respectively, both in
263 Replicate 1). A single adult Chaoborus sp. sampled from Replicate 1 on 22 May 2017 (Day 53) could
264 not be identified to species level. Therefore, of the successfully emerged adults, 99.68% were C.
265 obscuripes and only 0.24% were C. crystallinus.
266
267 Males appeared to emerge slightly earlier than females (Figure 1). In the first week of emergence 42
268 males were recorded compared with 8 females. Before 22 May there were generally more freshly
269 emerged males than females whereas the reverse was observed after this date.
270
271 Mean values for water temperature, dissolved oxygen, pH and conductivity measured weekly in each
272 microcosm are summarised in Table 3. Water temperature was measured every 30 minutes
273 throughout the study in one microcosm in replicate 1. The raw data for these environmental condition
274 readings are available in a data file “Environmental Conditions for Emergence study” and have been
275 summarised as daily maximum, minimum and mean values in Figure 2.
276
277 Figure 2: Daily maximum, minimum and mean water temperatures recorded in replicate 1
278 during the emergence study
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280 Table 3: Weekly water conditions during the Chaoborus emergence study
-1 Date Mean Water Temp. (°C) Mean Water pH Mean DO2 (%ASV*) Mean Cond. (µS cm )
measured (Standard Deviation) (Standard Deviation) (Standard Deviation) (Standard Deviation)
05-Apr 13.50 (0.24) 7.81 (0.04) 92.98 (8.54) 961.25 (9.18)
12-Apr 12.25 (0.13) 8.17 (0.09) 127.45 (18.54) 854.00 (16.02)
19-Apr 9.20 (0.23) 8.09 (0.11) 124.23 (7.23) 814.00 (5.72)
27-Apr 8.33 (0.10) 8.71 (0.12) 134.05 (2.82) 766.00 (6.98)
03-May 11.00 (0.14) 9.32 (0.07) 138.875 (2.17) 745.00 (4.55)
11-May 14.60 (0.61) 9.74 (0.01) 147.30 (4.20) 733.25 (6.34)
19-May 15.35 (0.10) 9.74 (0.05) 133.95 (0.83) 678.75 (8.18)
26-May 17.30 (0.32) 9.78 (0.14) 132.05 (7.27) 683.25 (9.57)
02-Jun 19.83 (0.33) 10.20 (0.35) 152.78 (19.52) 682.25 (10.69)
09-Jun 20.48 (0.26) 10.38 (0.15) 171.50 (6.88) 637.25 (11.73)
16-Jun 21.68 (0.15) 10.33 (0.02) 178.58 (14.48) 653.00 (11.92)
281
282 * ASV - Air Saturation Value
283
284 Voltinism
285 Daily mean water temperatures, pH, dissolved oxygen and conductivity values recorded during the
286 life cycle trial are presented in Table 5. The development of Chaoborus from the initial egg rafts
287 introduced into each unit are presented for each replicate in Figure 3. Egg rafts added to the first
288 microcosms of replicates 1, 2, 4 and 5 failed to establish at the first attempt and replicates 1, 2 and 5
289 were re-initiated at intervals as fresh egg rafts became available. The re-initiated replicates 1 and 5
290 both progressed through to a fourth generation. These two replicates were found to contain both C.
291 obscuripes and C. crystallinus in their respective first units although the larvae sampled from units 2 –
292 4 in both replicates were all C. crystallinus. In both replicates, C. obscuripes were only found in July,
293 while the specimens of C. crystallinus were only found in October. Replicate 4 could not be re-
294 initiated with egg rafts as none were available at a suitable time. Monitoring of this unit showed that
295 no larvae were present at any time, confirming the effectiveness of the systems in preventing
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296 immigration of Chaoborus spp. from outside. The re-initiation of replicate 2 produced only two
297 generations which, when sampled in October, were found to consist only of C. crystallinus.
298
299 Populations of Chaoborus in replicates 3 and 5 progressed through to a third and fourth generations
300 respectively. Larvae sampled from the first unit in July were found to be C. obscuripes, although
301 when re-sampled in October, both C. obscuripes and C. crystallinus were found to be present.
302 Generations 2 and 3, both sampled in October, were found to consist only of C. crystallinus. Replicate
303 6 also did not require re-initiation but as the second unit did not establish successfully, larvae were not
304 sampled from Unit 1 until October, in order to give the maximum opportunity for more egg
305 deposition to restart Unit 2. In practice, a second production of egg rafts did not occur in Unit 1 and
306 therefore, Unit 2 could not be re-started. Only six late-instar larvae remained in Unit 1 by the October
307 sampling and all were found to be C. obscuripes.
308
309 Minimum egg-to-egg times for C. crystallinus are summarised in Table 4 and ranged from 14 days
310 (Replicate 5, Unit 1) to 56 days (Replicate 3, Unit 2). As only C. crystallinus was found in the
311 second, third and fourth generations of any replicate, it is not possible to draw any conclusions
312 regarding the egg-to-egg timings for C. obscuripes. The shortest generation time of 14 days occurred
313 when the water temperature was at its highest (Fig. 3) in late June and early July. However, the
314 longest observed development time of 56 days also spanned this period so other variables such as prey
315 density are clearly involved in determining Chaoborus development rate.
316
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318 Table 4: Egg to egg development times of Chaoborus crystallinus
Unit 1 Unit 2 Unit 3
Dates / Duration (days) Dates / Duration (days) Dates / Duration (days)
Replicate 1 21 June-17 July (26) 17 July- 24 Aug. (38) 24 Aug-15 (22)
Sept*
Replicate 2 28 July - 29 Aug. (32) No successful second No successful third
generation generation
Replicate 3 8 May-23 June (46) 23 June - 18 Aug. (56) No successful third
generation
Replicate 5 21 June- 5 July (14) 5-28 July (23) 28 July - 24 (27)
Aug.*
Replicate 6 10 May-23 June (44) No successful second No successful third
generation generation
Mean 32.4 39.0 24.5
Overall mean 32.8 (S.D.13.0 days)
319
320 Although environmental conditions were recorded in all replicates during the study, they did not
321 differ significantly for any of the measured variables. The water temperature, dissolved oxygen levels,
322 pH and conductivity are presented in Table 5. The presence of the mesh enclosures had negligible
323 impact on water temperature in the microcosm. Temperatures in an open microcosm were compared
324 to those in an enclosed one and found to be very similar throughout the study.
325
326 Figure 3: Development of Chaoborus from introduced egg rafts in each microcosm that
327 sustained populations
328
329
330
331
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332 Table 5: Environmental conditions in microcosms in the life cycle study
Mean Mean STDEV STDEV of Mean DO2 STDEV of STDEV of Date Temp. Mean pH Cond. (n) of Temp pH DO2 Cond (°C) (%) (µS cm-1)
25-Apr 13.5 n/a 8.1 n/a 134.6 n/a 802 n/a 1 27-Apr 9.2 n/a 8.2 n/a 114.9 n/a 785 n/a 1 03-May 11.0 3.04 8.3 0.24 102.7 10.32 818 8.49 2 08-May 13.4 0.14 8.4 0.20 103.6 7.64 - - 2 10-May 16.8 0.35 8.5 0.13 107.7 13.15 - - 2 11-May 15.5 0.43 8.4 0.18 107.3 11.65 793 44.08 6 19-May 15.3 0.18 8.6 0.21 112.6 11.80 739 45.68 6 26-May 17.6 0.48 8.8 0.56 122.1 18.77 724 51.04 6 02-Jun 18.8 0.40 9.4 0.92 141.5 40.68 713 65.88 6 09-Jun 19.3 0.60 10.0 0.87 162.3 23.52 670 54.65 6 16-Jun 21.8 0.38 10.2 0.31 172.4 19.28 675 44.48 6 21-Jun 20.4 0.19 10.0 0.24 156.4 18.23 676 40.69 6 23-Jun 23.2 0.85 10.0 0.09 156.5 13.44 - - 2 28-Jun 17.7 0.12 10.1 0.34 156.1 19.33 661 40.05 8 07-Jul 25.2 0.49 10.2 0.28 196.7 32.63 674 49.94 7 14-Jul 19.2 0.27 10.3 0.22 170.4 18.59 688 109.07 10.00 21-Jul 21.3 0.20 10.2 0.26 205.4 19.18 661 42.34 9.00 28-Jul 19.0 0.30 10.1 0.39 160.5 26.67 627 42.30 12.00 02-Aug 18.3 0.56 10.14 0.47 165.2 26.30 645 54.24 12.00 11-Aug 19.5 1.02 10.12 0.44 156.9 20.84 601 40.65 12.00 18-Aug 19.9 0.39 10.00 0.42 163.2 14.88 599 46.70 12.00 24-Aug 19.4 0.07 9.8 0.39 130.8 45.33 - - 2 25-Aug 17.1 0.48 9.7 0.36 123.2 22.14 583 49.29 14.00 29-Aug 22.6 n/a 9.2 n/a 139.6 n/a - - 1 06-Sep 16.8 0.21 9.7 0.39 136.9 20.62 588 58.84 15.00 13-Sep 13.9 0.24 9.4 0.55 111.2 15.97 588 58.55 15.00 15-Sep 14.6 n/a 7.5 n/a 96.8 n/a - - 1 20-Sep 12.3 0.13 9.6 0.54 113.3 12.43 577 57.73 15.00 333
334 Discussion
335 Emergence of adult Chaoborus spp. from overwintered larvae in South Eastern England commenced
336 on 13 April 2017 (14 days after being introduced to artificial microcosms, Day 0) and peaked on 2
337 May 2017 (Day 33). The majority of emergence was completed by early June (the last occasion when
338 emergence of Chaoborus spp. occurred in all four replicates was 3 June 2017, Day 65). Of the 2027
339 fourth instar larvae introduced into the microcosms, the mean emergence success was 60.9%, ranging
340 from 51.4% in Replicate 4 to 66.2% in Replicate 1. As indicated in (22), it is likely that carnivory
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341 was responsible for the emergence success being less than 100%. All except four of the emergent
342 adults were C. obscuripes, which was consistent with the populations of Chaoborus spp. known to
343 inhabit the source pond in previous years. These results show that the emergence of adult C.
344 obscuripes originating from the pupation of post-overwintering fourth instar larvae took place over a
345 clearly defined period between mid-April and early June. Males appeared to emerge slightly earlier
346 than females (Figure 1). In the first week of emergence 42 males were recorded compared with 8
347 females.
348
349 The results of the voltinism study showed that C. crystallinus produced up to four discrete generations
350 within the experimental period. Since two replicate microcosm groups exhibiting four generations
351 were both re-initiated several weeks after the season’s egg deposition commenced, one more
352 generation may have been possible. There may be some differences in developmental times or
353 reproductive success between species since from the second generation onwards populations were
354 dominated by C. crystallinus, despite the presence of C. obscuripes in the first generations. It is
355 possible that the test units or their conditions may have been favourable for oviposition by C.
356 crystallinus but not by C. obscuripes. In "the wild" C. obscuripes is almost always associated with
357 larger bodies of water (large ponds, small lakes and reservoirs).
358
359 The time for development from egg-to-egg ranged from 14 days to 56 days with a mean of 32.8 days
360 (Standard Deviation 13.0 days) indicating a high degree of phenotypic plasticity in C. crystallinus life
361 history strategies. Given that environmental conditions within the microcosms were all very similar it
362 seems most likely that differing levels of prey density contributed to the wide range of development
363 times. Prey abundance and diversity was not determined as the influence of prey availability was
364 outside the scope of this study. However, this warrants further investigation as feeding is clearly a
365 significantly influential factor in development times.
366 This study confirmed that under temperate conditions C. crystallinus exhibits a multi-voltine life
367 history.
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